Project Summary: Although Juvenile Myoclonic Epilepsy (JME) is the most common form of inherited adolescent epilepsy, its underlying pathology remains poorly understood. Mutations in two genes that encode motile cilia structural proteins ? EFHC1 and EFHC2 ? have been shown to cause JME, providing the first genetic link between motile cilia and epilepsy. Motile cilia are microtubule-based cellular appendages that undulate repeatedly to move extracellular fluid. Outside of epilepsy, failure to generate extracellular fluid flow results in a variety of serious human disorders, including primary ciliary dyskinesia, hydrocephalus, and hearing loss. The maintenance of motile cilia structure is integral to cilia function, and the cilia biology field has a strong focus on understanding this complex interplay. For example, motile cilia must be able to bend to propagate a beat stroke, yet, they must also be stable enough to withstand the force generated by their own beating. Motile cilia beating relies on the microtubules that comprise them. The motile cilia axoneme consists of nine sets of modified doublet microtubules arranged radially around a central pair of microtubules. Cryo-electron tomography has revealed conserved densities within the lumen of the doublet microtubules that have been termed Microtubule Inner Proteins (MIPs). These densities are unique to ciliary axonemes, and their protein components and functions are currently unknown. It is likely that the loss of MIPs will affect the structural integrity of the motile cilia axoneme. Little is known about the JME-linked motile cilia proteins EFHC1 and EFHC2, which are microtubule-associated components of the ciliary axoneme. We have initiated investigations into the functions of EFHC1 and EFHC2 in the aquatic ciliate Tetrahymena thermophila (Tetrahymena). We discovered that the Tetrahymena orthologs of EFHC1 and EFHC2 ? Bbc73 and Bbc60, respectively ? are axonemal proteins required for the function of motile cilia beating. We were also excited to find that Bbc73 and Bbc60 are necessary for the formation of a number of MIPs located in the A-tubule of the axonemal doublet microtubules. We performed a mass spectrometry screen to identify proteins that require the EFHC proteins for their localization to ciliary axonemes in Tetrahymena. We identified a number of proteins of interest, including CAPS, a small calcium-binding protein that localizes to ciliary axonemes in a Bbc73- and Bbc60-dependent manner. The long-term goals of this project are: to understand the role of EFHC proteins in motile cilia function; to identify MIP components; and to determine the function of MIPs within axonemal doublet microtubules. To achieve these goals, we will: 1) determine the function and localization of EFHC proteins within motile cilia; 2) identify components of motile cilia axonemes that require EFHC proteins for their localization, or that are in close proximity to EFHC proteins; and 3) functionally characterize CAPS and other newly identified axonemal proteins that are potential MIP components. The results of our proposed experiments will answer fundamental questions about EFHC proteins in cilia biology and may provide novel insights into the pathology of epilepsy.